The specific aims of this proposal are: i) characterization of the molecular defects in ICF syndrome, and ii) the role of shifting replication borders in tissue-specific gene regulation. I. The ICF syndrome is characterized at the molecular level by DNA hypomethylation of certain heterochromatic regions including centromeric satellite II and the inactive X. Three questions will be considered: a) the identity of the defective gene in ICF which will furnish an important clue as to the control of genomic methylation; b) the effect on X inactivation of congenital hypomethylation of the inactive X; and c) the identity of genes whose regulation is disturbed by hypomethylation of centromeric satellite II regions. II. We have discovered that the position of the late-early replication border of Xq27-Xq28 differs by nearly a megabase between fibroblasts and lymphoid cells. The region that is late in fibrloblasts and early in lymphoid cells contains genes that are candidates for repression by late replication. We hypothesize that the replication borders in expressing tissues are shifted so as to place these genes in the transcriptionally permissive early domain. Such border shifting may have evolutionary implications for genome organization. We propose an extensive test of this model.